ABSTRACT Obesity is a major global health issue. According to a recent report in the Journal of the American Medical Association non-Hispanic blacks have the highest age-adjusted rates of obesity (49.5%) compared with Mexican Americans (40.4%), all Hispanics (39.1%) and non-Hispanic whites (34.3%). However, genetics cannot account for the epidemic proportions of obesity in recent history. Environmental toxicants impact epigenetic programs of stem cell differentiation. Adipogenesis is among the best developed of differentiation assays and the genetic program of adipogenic differentiation is well characterized. Many obesogens have been shown to epigenetically regulate adipogenesis. We propose to develop an easy-to-use cryopreserved obesogenesis assay plate for sale and distribution. This assay plate will serve as a key reagent in the development of high-throughput predictive models for identifying and measuring biological response in humans to 'obesogen' toxicants. The assay plate will be pre-seeded with adipogenesis-responsive luciferase reporter- transduced preadipocytes, cryopreserved, shipped to customers, rewarmed and then used for direct, rapid, reproducible and sensitive testing of potential obesogens. This technology will help to prioritize chemicals for more extensive toxicological evaluation, support more predictive models of in vivo biological response, and permit assessment of preadipocytes (derived from human inducible pluripotent stem cells from individuals with different genetic or disease backgrounds). The proposed work in this Phase SBIR I feasibility study will be performed in 2 specific aims. In the first aim preadipocytes will be stably transduced with an adipogenesis- responsive dual-luciferase reporter system consisting of the 'Firefly' luciferase test and 'Renilla' luciferase positive reporters. These clones will be challenged in a standard adipogenic assay using replicate serial dilutions and combinations of two established obesogens. In aim 2 optimization experiments for the cryopreservation, storage and thawing of transduced and control adherent preadipocytes on 96 well assay plates will be performed. The assessment methods will include 1) cell viability and proliferation, 2) base line adipogenic differentiation, and 3) response to obesogen(s). Feasibility for progression to Phase II will be demonstration of >70% viability, cell survival with retention of attachment to the tissue culture substrate, and functional adipogenesis-responsive reporter system post-thaw in 96 well plates. In Phase II we will assess a broad range of potential obesogens employing our initial transduced clones and additional preadipocyte clones from individuals with different diseases and genetic backgrounds. We will also assess other multi-well plate formats for even higher throughput applications and, if necessary, further modify adipogenesis detection systems to increase the obesogenesis assay sensitivity.